The present invention is related to antimicrobial peptides and methods of use thereof in vivo, and more particularly to substituted derivatives of peptide 20-44 of CAP37.
Antimicrobial therapies have advanced greatly over the years; however, people still die from infections and sepsis. The recent re-emergence of old infections once thought to be on the decline and the rapid evolution of resistant bacterial strains reinforces the critical need for designing and/or discovering new and more effective therapies. The most significant development in antibiotic therapy in the last decade has been the exploitation of a group of naturally occurring host proteins that are potent antimicrobials, to produce more effective, safer and broader acting drugs.
The immune system has at its disposal a number of mechanisms whereby it may protect itself from invading pathogens. The bactericidal killing mechanisms of the human neutrophil are particularly effective and comprise a collection of cationic granule proteins with potent antimicrobial action. One of these host-defense peptides is a novel, neutrophil protein known as CAP37 (Cationic Antimicrobial Protein of Molecular weight 37 kDa). The first isolation and purification of CAP37 was performed by Spitznagel and colleagues in 1984, who established that CAP37 had very strong antimicrobial activity mainly against gram-negative bacteria (1). Recently we demonstrated that in addition to the previously demonstrated antibiotic activity, CAP37 is a potent and highly specific chemoattractant for monocytes (2). We have determined the complete amino acid sequence of CAP37 (3) and cloned the gene for CAP37 (4). More recently we showed that CAP37 binds endotoxin (5).
Endotoxin is the outer membrane lipopolysaccharide (LPS) component of gram-negative bacteria. Bacterial LPS has very important clinical relevance because of its pleiotropic effect on various immune cells. It can evoke various disease symptoms ranging from chills and fever to circulatory collapse, multiorgan failure and death; a syndrome often referred to as endotoxic or septic shock. Despite aggressive treatment the mortality rates remain high, with septic shock being the most common cause of death in the intensive care unit and the thirteenth leading cause of death overall. Figures from the Centers for Disease Control in Atlanta, Ga. suggest that septic shock occurs at the rate of 175 per 100,000 people with the rate reaching almost 500 per 100,000 hospitalized patients. The death rate is often as high as 25 to 40%. Septic/endotoxic shock has been shown to be most often due to gram-negative bacteria although recent evidence would tend to indicate that the incidence of shock due to gram-positive bacteria and fungi is on the rise. Factors, that have contributed to the increasing incidence of sepsis include the new immunosuppressive therapies, invasive devices such as intravenous catheters and surgical devices and an aging population with many chronic diseases that predispose to sepsis.
The component responsible for the toxic effect of the LPS molecule is the lipid component called lipid A. This region is embedded in the outer membrane of the bacterium and believed to be reasonably constant between different species of gram-negative bacteria. The manner in which LPS evokes its lethal effects is by binding to cells such as monocytes, macrophages and/or endothelial cells, triggering them to produce toxic oxygen radicals, cytokines such as tumor necrosis factor xcex1 (TNFxcex1), various interleukins (IL-1, IL-6, and IL-8) and numerous other products. Our studies on CAP37 have led to the identification of a 25 amino acid peptide that mimics the antimicrobial and lipopolysaccharide binding functions of the native molecule (6). This synthetic peptide 20-44 based on residues 20 through 44 of native CAP37 has the amino acid sequence NQGRHFCGGALIHARFVMTAASCFQ (SEQ ID NO:1). This peptide has been tested in vitro and in vivo for antimicrobial activity as well as endotoxin binding activity (6). Peptide 20-44 shows strong in vitro bactericidal activity mainly against gram-negative bacteria such as Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa. In addition, the peptide is active against the gram-positive organisms Enterococcus faecalis and Staphylococcus aureus. In vivo experiments were conducted with peptide 20-44 in a conscious rat model of sepsis using purified endotoxin (5). Intravenous infusion of peptide 20-44 (3.0 mg/kg body weight), with Escherichia coli LPS (250 xcexcg/kg over 30 min) into conscious unrestrained rats prevented LPS-induced hyperdynamic and hypodynamic circulatory shock. Peptide 20-44 (0.2, 1.0, and 5.0 mg/kg) administered intravenously to conscious actinomycin-D sensitized rats following a lethal dose of LPS neutralized LPS toxicity, resulting in dose-dependent 7-day survialc rates of 30, 50 and 80% respectively. Peptide 20-44 (5.0 mg/kg) significantly inhibited the endotoxin-induced increase in circulating TNFxcex1 in sensitized rats. These data demonstrate that peptide 20-44 has the capacity to abolish in vivo biological responses to LPS that are relevant to human sepsis and to significantly neutralize the toxicity of circulating Escherichia coli LPS.
The results described above, as well as further information regarding CA37 peptides and their function are shown in U.S. Pat. Nos. 5,484,885; 5,458,874; 5,607,916; 5,627,262; and 5,650,392, and pending U.S. Ser. No. 08/840,519, the specifications of all of which are hereby incorporated herein by reference in their entireties.
The present application describes new peptide analogs (e.g., see FIG. 1) based on the native sequence of CAP37 peptide 20-44 or 23-42 and their use as effective therapeutics against certain bacterial infections and diseases caused by bacterial infection.
In a particularly preferred embodiment, the present invention contemplates a peptide, and a composition comprising said peptide, which is a derivative of CAP37 peptide 23-42 (SEQ ID NO:2) wherein one of the cysteine residues at positions 26 and 42 is substituted with a serine or threonine residue and one of the cysteine residues at positions 26 and 42 is left unsubstituted. Further, the peptide derivative may comprise at least one of the following substitutions: phenylalanine replaced by tyrosine; glycine replaced by alanine; valine replaced by alanine, leucine, or isoleucine; alanine replaced by leucine, isoleucine or valine; leucine replaced by alanine, isoleucine or valine; isoleucine replaced by valine, leucine or alanine; serine replaced by histidine, arginine, or lysine; and threonine replaced by serine.
As noted elsewhere herein, the peptide derivative may be a derivative of CAP 37 peptide 20-44 modified as described above for the derivative of CAP37 peptide 23-42 above.
The invention further comprises a method of treating a bacterial infection or septic shock in a patient, or prophylactically preventing septic shock in a subject comprising administering a therapeutically effective amount of a peptide derivative claimed and described herein.
Further, the peptide derivative contemplated herein may comprise a peptide (SEQ ID NO: 59 and SEQ ID NO: 60) having the formula:
R-H -X1-X2-X3-X4-X5-X6-X7-H-X8-R-X9-X10-M-X11-X12X13-X14; -X15
wherein X1 and X9 are phenylalanine and/or tyrosine; X2 and X15 are cysteine, serine, and/or threonine; X3 and X4 are glycine and/or alanine; X5-X8, X10, X12 and X13 are alanine, leucine, isoleucine and/or valine; X11 is serine and/or threonine; X14 is serine, threonine, histidine, arginine or lysine; R is arginine; H is histidine; M is methionine; and with the proviso that one of X2 and X15 is cysteine and one of X2 and X15 is serine or threonine.
The present invention further comprises a DNA molecule having a nucleotide sequence encoding a peptide having an amino acid sequence as defined in any of the amino acid sequences listed or described herein, in particular, those having substituted cysteine residues at positions 26 or 42.